Methods and compositions for detecting esophageal neoplasias and/or metaplasias in the esophagus
Inventors
Markowitz, Sanford D. • Moinova, Helen • Chak, Amitabh • Willis, Joseph • LaFramboise, Thomas
Assignees
Case Western Reserve University
Publication Number
US-12258632-B2
Publication Date
2025-03-25
Expiration Date
2037-07-05
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
The disclosure provides methods for identifying genomic loci (e.g., vimentin and/or SqBE18) that are differentially methylated in metaplasias (e.g., Barrett's esophagus) and/or neoplastic cancers (e.g., esophageal cancers). Identification of methylated genomic loci has numerous uses, including for example, to characterize disease risk, to predict responsiveness to therapy, to non-invasively diagnose subjects and to treat subjects determined to have gastrointestinal metaplasias and/or neoplasias.
Core Innovation
The invention provides methods for identifying genomic loci, specifically SqBE18, that are differentially methylated in metaplasias such as Barrett's esophagus and neoplastic esophageal cancers. By obtaining an esophageal sample from a subject, treating it with bisulfite, amplifying bisulfite converted SqBE18 DNA, and analyzing the fraction of methylated reads, the invention enables the detection of methylated DNA associated with esophageal neoplasias or metaplasias.
The problem being addressed is the deficiency in current methods for detecting esophageal neoplasias or cancers, which are either invasive, uncomfortable, expensive, or have issues with sensitivity and specificity. Traditional diagnostic methods, such as endoscopy or imaging, may yield false positives/negatives and are not ideal for early detection of esophageal neoplasias or their precursor lesions.
The core innovation lies in the molecular detection of methylation at informative loci—particularly SqBE18—using bisulfite conversion and next-generation sequencing to quantify methylated CpG dinucleotides in DNA from esophageal samples. If the portion of methylated reads exceeds a defined threshold (such as at least 0.1%), the subject is determined to have esophageal neoplasia or metaplasia and can be directed or selected for appropriate therapy, such as cryotherapy, ablation, surgery, or administration of specific chemotherapeutic agents.
Claims Coverage
The patent claims encompass multiple inventive features addressing the detection and treatment of esophageal neoplasia or metaplasia using methylation analysis of SqBE18 loci.
Detection and treatment based on methylation of SqBE18 in esophageal samples
A method that involves: 1. Providing an esophageal sample from a subject. 2. Treating the sample with bisulfite to obtain bisulfite converted SqBE18 nucleic acid sequences. 3. Amplifying the bisulfite converted sequences to generate amplicons (reads) that are at least 90% identical to SEQ ID NOs: 8318, 8360, 8332, and/or 8374, or a fragment of at least 50 nucleotides. 4. Classifying a read as methylated if at least 70% of the CpG cytosines in the read are methylated. 5. Calculating the percentage of methylated reads, and if at least 0.1% of total reads are methylated reads, administering a treatment (cryotherapy, photodynamic therapy, radiofrequency ablation, laser ablation, argon plasma coagulation, electrocoagulation, stenting, surgery, or specified therapeutic agents) to the subject.
Detection of methylation status by next-generation sequencing using specified primers and amplicons
The invention specifies: - Using next-generation sequencing to determine sequences of bisulfite converted DNA. - Amplified portions may cover 21 CpG dinucleotides present in native SqBE18. - Primers used for amplification can include SEQ ID NOs: 8388 and/or 8402. - The amplicons comprise sequences at least 90–95% identical to SEQ ID NOs: 8318, 8360, 8332, and/or 8374.
Quantitative thresholds for classifying methylated samples for diagnosis or treatment
The claims define multiple thresholds for classifying an esophageal sample as 'methylated' based on the percent of methylated reads detected: - Thresholds include as low as 0.1%, and ranges such as 0.5% to 3.5%, 1% to 3.11%, and at least 3.11% methylated reads. - Meeting the threshold classifies the sample as methylated and determines subject eligibility for specific interventions.
Diagnosis and clinical management by methylation analysis and follow-up endoscopy
A method for detecting methylation status in an esophageal sample includes all the analytic features above, and for samples meeting threshold methylation, the subject is classified as having esophageal neoplasia or metaplasia and may be selected for an endoscopy.
The claims collectively cover methods for detecting and quantifying methylation of SqBE18 loci in esophageal samples to diagnose neoplasia or metaplasia, and using this molecular information to guide diverse therapeutic strategies, including surgery, ablation, and chemotherapy.
Stated Advantages
The methods provide increased sensitivity and specificity for detecting esophageal adenocarcinoma, Barrett's esophagus, and related neoplasias compared to traditional diagnostic approaches.
The invention allows for non-invasive or minimally invasive sample collection (such as brushings or balloons), reducing discomfort, risk, and expense compared to conventional endoscopy.
Quantitative molecular assays enable early detection and monitoring of disease progression, facilitating timely intervention and tailored treatment based on precise methylation thresholds.
The approach enables objective and reproducible diagnosis, minimizing false positives and negatives associated with existing imaging or tissue sampling methods.
Documented Applications
Non-invasive or minimally invasive diagnosis of esophageal neoplasia, metaplasia, or Barrett's esophagus through detection of methylated SqBE18 loci in esophageal samples.
Guiding clinical management and selection of treatment modalities, such as cryotherapy, photodynamic therapy, ablation, stenting, endoscopic mucosal resection, esophagectomy, anti-reflux surgery, or chemotherapy, for subjects with methylation-detected disease.
Identifying subjects for follow-up endoscopy based on methylation status of esophageal samples.
Monitoring response to therapy and disease progression or regression by serial assessment of methylation in esophageal tissue or brushing samples.
Interested in licensing this patent?